Long lasting immunity in trichinellosis - insight from a small study group.

Trichinellosis in humans is most often caused by the parasite Trichinella spiralis. The clinical course of the disease is diverse and the symptoms can vary from mild to severe. Symptoms usually disappear within a few months, but encysted larvae in the muscles can cause myalgia and weakness that last for years. However, the existence of chronic trichinellosis as a disease is still debatable. This study presents the results obtained at the National Reference Laboratory for Trichinellosis - INEP, Serbia. The study was conducted to assess the immunoserological, biochemical and symptomatic disease parameters of twelve patients who acquired trichinellosis 13 and 18 years ago, respectively. They were involved in two T. spiralis outbreaks in Serbia, at the village of Kumane and the city of Belgrade (nine and three patients, respectively). Results indicated the presence of specific anti-Trichinella antibodies in 83% of the total number of patients. However, while the humoral immune response to Trichinella lasted for more than a decade reaching almost two decades after the acute infection phase (7/9 and 3/3 respectively, in two outbreaks), persistence of chronic muscular pain, as the most prolonged symptom of trichinellosis, could be found in the majority of patients from the Kumane outbreak (7/9). As a consequence, these patients suffered from limitations in daily living activities for the same period of time. The results presented in this paper are our contribution to the view that trichinellosis as a chronic disease with symptoms exists and may be related to the severity of the disease in the acute phase.

A competitive enzyme-linked immunosorbent assay for rapid detection of antibodies against Trichinella spiralis and T. britovi - one test for humans and swine.

Infection with parasites from the Trichinella genus occurs in many vertebrates but disease only occurs in humans (trichinellosis). Humans are infected after the consumption of raw or undercooked meat from infected wild or domestic animals (usually swine or horses). Using the monoclonal antibody (mAb) 7C2C5, specific for an epitope unique to the muscle larvae of the genus Trichinella, we have developed a competitive enzyme-linked immunosorbent assay (c-ELISA) that enables the rapid detection of Trichinella-specific antibodies in sera originating from two different host species (human, swine) infected with either Trichinella spiralis or Trichinella britovi. This novel c-ELISA exhibited 100% specificity and sensitivity, as confirmed by a Western blot test. The assay is easy to use (one incubation step), and the time required for the procedure (45 min) is shorter than in any other ELISA format. This test could be useful for both the detection and surveillance of Trichinella infections.

Immunomodulatory potential of particular Trichinella spiralis muscle larvae excretory-secretory components.

Excretory-secretory antigens of Trichinella spiralis muscle larvae can induce the semi-matured status of rat dendritic cells. This may at least partly be the consequence of transient activation of extracellular signal-regulated kinases 1/2 (ERK1/2). Here we investigated the potential of several components of excretory-secretory antigens (native fraction containing 45, 49 and 53kDa proteins and recombinant Tsp53, representing one of the constituents of this fraction) to demonstrate previously observed effects of excretory-secretory antigens on dendritic cells in vitro, characterised by establishment of a particular phenotype (very low MHC II expression, moderate CD86 expression and significant ICAM-1 expression) and functional properties (low production of pro-inflammatory cytokine IL-12p70, and high production of IL-10 and TGF-ß). Dendritic cells activated by these components were able to provoke proliferation of naïve T cells and their polarisation towards Th2 and anti-inflammatory responses. The investigated antigens had almost the same capacity to induce IL-4 and IL-10 production from T cells as excretory-secretory antigens, but failed to induce significant TGF-ß synthesis. It could be concluded that the investigated excretory-secretory antigens components can largely reproduce the immunomodulatory effects of the complete excretory-secretory antigens and therefore may be considered as molecules important for creation of the anti-inflammatory milieu achieved by the parasite.

Application of dendritic cells stimulated with Trichinella spiralis excretory-secretory antigens alleviates experimental autoimmune encephalomyelitis.

The parasitic nematode, Trichinella spiralis (T. spiralis), exerts an immunomodulatory effect on the host immune response through excretory-secretory products (ES L1) released from encysted muscle larvae. Our model of combined T. spiralis infection and experimental autoimmune encephalomyelitis (EAE) in Dark Agouti (DA) rats demonstrated a significant reduction in EAE severity in infected animals. Recently, we have created an immune status characteristic for the live infection by in vivo application of dendritic cells (DCs) stimulated with ES L1 products of T. spiralis muscle larvae. Moreover, these cells were able to ameliorate EAE when applied 7 days before EAE induction. ES L1-stimulated DCs increased production of IL-4, IL-10 and TGF-ß, and decreased production of IFN-γ and IL-17, both at the systemic level and in target organs. A significant increase in the proportion of CD4+CD25+Foxp3+ T cells was found among spleen cells, and CNS infiltrates from DA rats treated with ES L1-stimulated DCs before EAE induction, compared to controls injected with unstimulated DCs. Regulatory T cells, together with elevated levels of IL-10 and TGF-ß, are most likely involved in restraining the production of Th1 and Th17 cytokines responsible for autoimmunity and thus are responsible for the beneficial effect of ES L1-educated DCs on the course of EAE. Our results show that ES L1 antigen-stimulated DCs are able not only to provoke, but also to sustain anti-inflammatory and regulatory responses regardless of EAE induction, with subsequent amelioration of EAE, or even protection from the disease.

The impact of Trichinella spiralis excretory-secretory products on dendritic cells.

Parasitic nematode Trichinella spiralis exert immunomodulatory effect on the host immune response through excretory-secretory products (ES L1) released from the encysted muscle larvae. Rat bone-marrow derived dendritic cells (DCs) stimulated with ES L1 antigens acquire semi-matured status and induce Th2 and regulatory responses in vitro and in vivo. Priming naïve T cells in vitro with ES L1 pulsed DCs caused strong Th2 polarization, accompanied by elevated production of regulatory cytokines IL-10 and TGF-ß and no increase in the proportion of CD4+CD25+Foxp3+ among the effector T cell population. In vivo T cell priming resulted in mixed Th1/Th2 cytokine response, with the dominance of the Th2 type and elevated levels of regulatory cytokines. Significant increase in the proportion of CD4+CD25+Foxp3+ cells was found among recipient's spleen cells. We have achieved to create immune status characteristic for the live infection by in vivo application of DCs educated with ES L1 antigens.

Trichinella spiralis antigens prime mixed Th1/Th2 response but do not induce de novo generation of Foxp3+ T cells in vitro.

Many parasitic helminth infections induce Th2-type immune responses and engage the regulatory network. In this study, we specifically investigated the influence of antigens derived from different life stages of the helminth Trichinella spiralis on the polarization of naive CD4(+) T cells by dendritic cells. Results obtained from C57BL/6 mice showed that T. spiralis derived antigens have the capacity to induce bone marrow-derived dendritic cells to acquire an incompletely mature phenotype that promotes a significant proliferation of naive CD4(+) T cells and a mixed Th1/Th2 cytokine profile with the predominance of Th2 cytokines. Increased production of IL-4, IL-9, IL-10 and IL-13 accompanied increased IFN-γ. Furthermore, dendritic cells pulsed with T. spiralis antigens did not induce an increase in the population of Foxp3(+) T regulatory cells. Although other helminth antigens have demonstrated the capacity to induce de novo generation of Foxp3(+) T regulatory cells, here our in vitro studies provide no evidence that T. spiralis antigens have this capacity.

Mechanisms of modulation of experimental autoimmune encephalomyelitis by chronic Trichinella spiralis infection in Dark Agouti rats.

Trichinella spiralis is a helminth that provokes Th2 and anti-inflammatory type responses in an infected host. Our previous studies using Dark Agouti (DA) rats indicated that T. spiralis infection reduced experimental autoimmune encephalomyelitis (EAE) severity in rats. The aim of this study was to analyse the mechanisms underlying EAE suppression driven by T. spiralis infection. Reduced clinical and histological manifestations of the disease were accompanied by increased IL-4 and IL-10 production and decreased IFN-gamma and IL-17 production in draining lymph node cells. This indicates that T. spiralis infection successfully maintains a Th2 cytokine bias regardless of EAE induction. High IL-10 signifies parasite-induced anti-inflammatory and/or regulatory cell responses. Transfer of splenic T cell-enriched population of cells from T. spiralis-infected rats into EAE immunized rats caused amelioration of EAE and in some cases protection from disease development. This population of cells contained higher proportion of CD4(+) CD25(+) Foxp3(+) regulatory cells and produced high level of IL-10 when compared with uninfected rats.

Strain difference in susceptibility to Trichinella spiralis infection between dark agouti and albino oxford rats.

The influence of host genetics on the susceptibility to primary Trichinella spiralis infection has been extensively studied in a mouse model, but has not been clarified for rats. Analyses of interstrain and intrastrain genetic variation in response to infectious agents could be beneficial not only for elucidating the genetic basis of host resistance/susceptibility, but for revealing immune response mechanisms as well. The aim of this study was to analyse interstrain differences in worm burdens and cytokine production between Albino Oxford (AO) and Dark Agouti (DA) rats in muscle phase of T. spiralis infection. Clear strain-dependent variation was observed in the number of T. spiralis larvae per gram (lpg) of muscle tissue where values for DA rats (626.7+/-171lpg) vastly exceeded those found in AO rats (49.8+/-25.9lpg, p<0.001). Differences between the strains were also noticed in key cytokine levels. In infected AO rats, the cytokine production remained in favor of Th1 type response, while infected DA rats showed a shift towards a Th2 type response. The level of regulatory IL-10 was significantly increased only in T. spiralis infected DA rats. Our results provide evidence that DA rats express higher susceptibility to T. spiralis infection in comparison to AO rats with respect to muscle larvae burden. The infection in DA rats was accompanied by the production of anti-inflammatory cytokines, while the response of AO rats was characterized by a proinflammatory type of immune response.

Characterization of rat bone marrow dendritic cells initially primed by Trichinella spiralis antigens.

Pathogen-derived products have the capacity to induce maturation of bone marrow-derived dendritic cells (BMDCs)into populations of effectors cells that polarize Th cells toward Th1 or Th2 phenotype via different mechanisms. Since those mechanisms are not entirely clear for helminths, and almost completely unknown for Trichinella spiralis(TS), we started an investigation of the effects of TS antigens (four different antigens isolated from all three life-cycle stages of parasite)on maturation of BMDCs and their potential to present TS antigens. The expression of MHC class II, costimulatory molecules CD86, CD54, IL-10 and IL-12p70 cytokine production were measured after 2 days of BMDCs cultivation with TS antigens. While parasitic antigens did not significantly alter the expression of MHC II, most of them, except crude muscle larvae antigens, up-regulated the expression of costimulatory molecules. BMDCs, primed with all TS antigens, released increased amounts of IL-10 and decreased amounts of IL-12p70. BMDCs, primed with TS antigens, induced significant proliferation of syngeneic TS sensitized lymph nodes cells and also stimulated the production of IL-4 by T cells purified from of TS infected DA rats. The results indicate that TS stimulated BMDCs leads to the polarization of the immune response towards regulatory and Th2 type.

Trichinella spiralis: modulation of experimental autoimmune encephalomyelitis in DA rats.

Helminth infection has a potent systemic immunomodulatory effect on the host immune response, which also affects the development of autoimmune diseases. We investigated the dose-dependent influence of Trichinella spiralis infection on experimental autoimmune encephalomyelitis (EAE). Our model of concomitant T. spiralis infection and EAE demonstrates that established infection of Dark Agouti (DA) rats with the parasite causes amelioration of the clinical course of induced EAE in a dose-dependent way. Infection with T. spiralis L1 stage muscle larvae (TSL1) reduced the severity of the autoimmune disease as judged by lower maximal clinical score, cumulative index, duration of illness and degree of mononuclear cell infiltration in T. spiralis infected animals compared to control, EAE-induced group. This study provides a valuable model of worm infection to investigate helminth-induced regulatory mechanisms for optimal benefit to the host.

The involvement of the macrophage mannose receptor in the innate immune response to infection with parasite Trichinella spiralis.

The macrophage mannose receptor (MR) is a pattern recognition receptor of the innate immune system that binds to microbial structures bearing mannose, fucose and N-acetylglucosamine on their surface. The MR can mediate endocytosis and phagocytosis, as well as activation of macrophages and antigen presentation. Since Trichinella spiralis antigens are rich in oligomannose residues, we investigated whether a mannose-recognizing receptor, such as the MR, participated in the host-parasite interaction. The results show that the MR (either on the surface of macrophages or in the purified form) recognizes and binds components of T. spiralis muscle larvae. The presence of parasites provoked activation of peritoneal macrophages, which was indicated by down-regulation of MR expression, and the stimulation of NO secretion. In vitro stimulation of macrophages with T. spiralis components resulted in increased NO and IL-6 production. However, while the MR was partially involved in stimulation of NO production, it did not mediate IL-6 secretion.

Anti-Trichinella antibodies detected in chronically infected horses by IFA and Western blot, but not by ELISA.

In the Balkan countries, where trichinellosis is a re-emerging zoonosis, it is of great importance to determine Trichinella infection prevalence among the major hosts, including horses. One method for monitoring prevalence is serological surveillance; however, the validity of serological methods in horses is not well understood. The dynamics of anti-Trichinella IgG production and circulating excretory/secretory (ES) antigens were investigated in three horses experimentally-infected with Trichinella spiralis. Horses were slaughtered at 32 week post infection (p.i.). Low worm burdens were found in all three animals. Anti-Trichinella IgG was detected up to 32 weeks p.i. by an indirect immunofluorescence assay (IFA) and by Western blot (Wb), but not by ELISA. The ELISA test detected antibodies for only a short period of time (up to 18 weeks p.i. using ES antigen or up to 20 weeks p.i. using tyvelose-BSA antigen). The presence of circulating muscle larvae ES antigen in sera of infected horses was observed by dot blot from the 4th week p.i. up to the 32nd week p.i.